Milestone 1: Preliminary design / State of the art
State of the art
Along with the development of 2D/3D printed polymers, there have been efforts for printing living cells or other organic molecules. There are various methods used, but the one of interest for the current project is modifying an inkjet printer into a bio/cell printer. Multiple scientists around the world have successfully modified commercial thermal and piezoelectric inkjet printers to accomplish this task [3]. The type of printed molecules vary from proteins or other organic molecules to mammalian living cells.
Drop-on-demand bioprinting is commonly used with modified desktop inkjet printers. Modification of desktop printers is both an advantage and disadvantage of this technique. The hardware interfacing with the printhead already exists and has been quality tested by the manufacturer. However, this hardware is designed to print on paper and contains numerous checks to prevent the printer from performing outside its normal specifications. Depending on the bioprinting application, the printer hardware must either be reverse-engineered or modified to bioprint. A printer can be modified to bioprint by using a bioprinting stage on one side of the printer and feeding paper through the other side to bypass the paper checks. Reverse engineering provides control of individual inkjet channels which is extremely beneficial for studying the fundamentals of inkjet bioprinting, especially with regard to channel throughput and clogging. However, reverse engineering a desktop printer may be beyond the capabilities of research groups that do not focus on engineering. Therefore, several groups have used the existing printer hardware with modifications to facilitate bioprinting.
One of the major modifications made to desktop inkjet printers is the addition of a Z axis in the paper tray. Since a desktop printer only prints two-dimensional sheets of paper, stacking multiple layers at once requires an additional mechanism to lower the printing area with each successive layer. This is typically performed by adding an electronic elevator to the paper tray that lowers upon receiving a signal from the operator to print another layer. In this way, additional layers can be stacked upon each other to create a three-dimensional construct from two-dimensional layers.
Despite the numerous applications of inkjet bioprinting, there are two major issues that prevent single-cell inkjet bioprinting from reaching its full potential. The first issue is the limited mobility of the printhead if no reverse engineering is performed. In the normal configuration the printhead is limited to less than 250mm on the X axis and the length of the printhead on the Y axis. Thus, only biological structures of limited size can be created with this type of bioprinting system. Second, inkjet cartridges suffer from low throughput. This is mainly due to deposition of salts in the microfluidic channels during the printing process. This often occurs when evaporation of water from the bio-ink drop leaves behind solid salts that block the channel orifice. Once the channel is clogged, it is virtually impossible to restore full functionality to that channel. Furthermore, cellular debris and other contaminants can clog the microfluidic chambers as well. As a result, inkjet cartridges typically can only print 400,000 cells per cartridge before failure. This throughput is too low to produce large tissue constructs.
It should be noted that older printers tend to produce better results after the modification, as they use ink cartridges with larger diameter nozzles, which do not clog easily and allow to print larger cells. In addition, older printers use mechanical paper feed sensors that are easier to bypass. (Printers with optical sensors can also be “tricked”). Similar attempts have been made using HP DeskJet 500 [4]. Each application of bioprinting requires a separate set of cell types and matrices. Studies of cell viability in thermal inkjet printheads have shown cell survival to be 70–90%, although some groups have determined that cells require a recovery period after bioprinting to restore membrane integrity.
Design Specifications
Printer: HP DeskJet D4260
Ink Cartridge specifications:
Black: No. 339 (21 ml) Drop: 15 picoliters Nozzles: 672 Nozzle diameter: Head Length: 0.5625 in DPI : max 1200 , fast 300, normal 600
Color: No. 343 (7 ml per color) Drop: 5 picoleters Nozzles: 600 Nozzle diameter ~= 10μm approx Head Length: 0.5 in DPI: max 1200, fast 300, normal 600
=====================================
Printer: HP DeskJet 825c
Ink Cartridge specifications:
Black: No. 15 (21 ml)
Drop: 33 picoliters
Nozzles: N/A
Nozzle diameter: ~35 μm (measured, not provided)
Head Length: ~0.5 in
DPI : max 600 , normal 300
Color: No. 17 (7 ml per color)
Drop: 9 picoliters
Nozzles: N/A
Nozzle diameter N/A (not measured, not provided)
Head Length: N/A
DPI: max 1200, normal 600
================================
SELECTED
Key Functions
Protocol for modifying the ink cartridge: Use any long tool with flat edge, for example a straight screwdriver (img 3 for explanations)
Remove any tapes/stickers on the cartridge exterior (img 1)
Remove the metal side panels. Use the tool you have selected as a lever by inserting it from the existing hole (img 2,3). After that it should look like image 4. We need the right panel, don't throw it away.
Punch a small hole on the aluminum sheet. There is ink inside so place under running water in a sink until no ink comes from the hole.
Remove both the aluminum sheets. They are glued in the border of the plastic cover. Using a sharp tool (i used the same screwdriver) you must pull it off. Inside there is a spring, which is not needed, and should be disposed.
Afterwards, remove the two metallic filters near the printing head. Using tongs will make the job easier.
Last step, is removing the soft, light colored plastic inside of the plastic cover. Its quite tricky, but can be done with the same screwdriver. Its edge should be placed between the dark and the light colored plastic. Near the printing head, there is a small button, which is an extension of the light plastic. You must use a knife/saw to cut it from the inside, otherwise the light plastic can't be removed.
Now your ink cartridge is ready to be cleaned and sterilized.
Step 1: Remove any stickers
Image 2:The entry point for the removal of the side panels
Image 3: Why you need a flat edge tool
Image 4: How it looks after the removal of the panels
Image 5: One of the aluminum sheets is removed. The metallic filter can be seen (red arrow). The inside is symmetrical, so the same components are on the other side
Image 6: The spring that can be disposed of. it lies between the two aluminum sheets
Protocol for cleaning ink cartridge:
The ink cartridge should be cleaned before and after use.
Fully submerge the cartridge in a beaker full of de-ionized water, and sonicate for 15 minutes before and after printing.
After sonication, remove the cartridge from the water, and shake out excess water.
Spray 70% ethanol into the cartridge to create a more aseptic environment. (!Note: Ensure that the ethanol has dried before adding bioink-cells)
Protocol for cleaning the printer:
Leave the printer body under UV light for at least 20min
Spray 70% ethanol on any surface, and especialy the ink holder
Protocol for printing samples:
Clean the printer body and the ink cartridge
Prepare your sample pattern in a text editor (e.g. Microsoft Word)
Connect your printer to your computer and the power supply. Connect the power supply of the Arduino micro-controller!!
Set the printer power on and wait for the machine preparations (~30 sec). The end will be defined by the positioning of the ink carriage on the right of the body and the halting of movement. Upper green light should be constantly lit
Set the appropriate printing surface
Appropriate dimensions:
Length: <10 mm (strictly!)
Width : =< 210 mm (with the value depending on the border option inside the text editing software
Thickness: =<500 mm
Supply your ink cartridge with your cell solution and place it in the ink carriage
Send print command from the computer
Wait until the ink carriage returns at its original position
You are ready to remove the printing surface
In order to repeat the process re-cleaning of the printer body (especially when printing different cell types) is highly advised and re-cleaning of the ink cartridge is mandatory
Milestone 2: Detailed design
Reverse engineering of a similar printer
We have deconstructed an Officejet J6400 All-in-One printer in order to discover the key mechanisms of the device, and which of those will or could be used for the task we want to accomplish. Both the J6400 and the D4260 use the same ink cartridges and it is quite possible to have the same mechanisms and control units. What we found is that the different mechanisms are:
1) Ink cartridges/print head holder
2) Paper feeding mechanism
3) Print head dust/air contact protection mechanism
4) Print head cleaning/unclogging mechanism
5) Paper feed sensor (mechanical lever & opto switch) (easy to bypass)
6) Lid status (open/close) sensor (mechanical lever & opto switch) (easy to bypass)
7) Paper and Print head carriage motors
8) Paper motor and print head carriage position feedback and sensors
Print head size & structure using microscope for D4260
In order to determine the size of cells that can be "printed" using the Deskjet D4260 printer, it is essential to know the diameter of the nozzles in each of the ink cartridges (blank/color). To do so, we disassembled the print head of an ink cartridge (typically a thin, silver foil with the nozzle holes and ink channels drilled in it) and examined it using two different microscopes.
According to the 3rd picture, the nozzle diameter is about 10μm with a 80μm spacing between nozzles. Only the tri-color ink was disassembled and inspected in detail. The nozzles of the black ink are expected to be slightly bigger because its drop volume is three times larger than the one of the tri-color ink (see ink specs.), but still it should not exceed 15μm. This allows only the printing of bio-molecules, prokaryotic cells and only small eukaryotic cells with average cell diameter roughly equal to the one of the nozzle.
Ink cartridges contain a sponge which absorbs the main volume of the ink. Between this sponge and the channels that lead the ink to the printing nozzles are placed various filters to keep solid particles from entering and blocking the nozzles. The pore diameter of the main sponge is smaller than 50μm, while that of the filters is way smaller than 10μm. These pores do not allow the ink cartridge to be filled with a cell solution, because most cells can not pass through and the filters will be quickly blocked. Furthermore, cleaning and sanitizing the cartridge is more difficult if it contains filters. As a result, both the sponge and the filters must be removed, before any other process is initiated.
Another important parameter is the minimum volume of bio-ink required. If the volume of ink is smaller that this quantity, the pressure generated from the inks own weight is not enough to overcame the hydraulic resistance of the channels. As a result, the ink never reaches the nozzles and the printing has very low quality or is not done at all. This parameter can only be measured by experimentation. For this printer its value is 0.5 ml.
Black Ink, Nozzles (circles) and Channels (lines)
Tri-Color Ink, Nozzles (circles) and Channels (lines)
Print head size & structure using microscope for 825c
The measurement was repeated for the black ink of the DeskJet 825c printer, which was also available for experimentation. As we can see on the left image, the nozzle diameter is 35μm, three times larger than that of the D4260, thus allowing the printing of much larger cells. The distance between successive nozzles is about 50μm with an 10μm offset from the straight, central line.
As for the minimum volume of ink of this printer, it was measured with experiments and it is 0.25 ml.
Black ink, print head detail
Black ink, nozzle diameter measurement
Other possible printers - Final Decision
The nozzle diameter of the printer's cartridges disallows most eukaryotic cells to be printed without serious stress. So we had to find printers that use older cartridges with larger nozzles. One of the most suitable options is the HP 26 Black Ink Cartridge [2.1] with a drop volume of 140-150 picoliters (>10 times larger than the HP 339 Ink) and a nozzle diameter of about 50μm. Unfortunately, most printers that use this cartridge are not sold today. Although, there are some used ones available but their price ( ~90$) exceeds the budget of this project. Additionally, it is unclear if these printers will still work during the lifetime of the project and whether spare parts and cartridges will be marketable the following years.
There are also some custom made print heads, usually controlled by an arduino device. They offer very low DPI values and accordingly large nozzle diameters. One of these is the InkShield with 96x96 DPI and nozzle diameter equal to 85μm, features that make it ideal for the purpose. The price of an InkShield DIY Kit is about 60$ [2.2], which is too beyond the project's budget.
To summarize, there are some good but limited options available in the market, but their price is the main reason we will choose to continue working with an already owned printer and setting an upper limit on the diameter of cells that can be printed. Both printers, D4260 and 825c, were reversed engineered and reached a level, where they could be manually operated to print cells or biomolecules. It was important, though, to choose only one of them for further experiments, automatize its printing process and modify it, until it reaches the desired design level. The main factor that affected this choice was the nozzle diameter and the range of cells that can be printed. Thus, the DeskJet 825c is considered, as previously mentioned, a superior choice, with the following advantages:
three times larger nozzle diameter
easier to use and reverse-engineer ink cartridges
smaller minimum volume of bio-ink required
simple design
larger space available for items that will be used as printing surfaces
Sensors and systems to be tricked / used
Paper feed sensor opto switch (and some thoroughly cleaned fingers)
Overview of the printer, sensors' positions
Rotational photosensor paired with the paper axis
Encoder for the rotational photosensor
Determining the sequence of functions during the printing
In order to modify the printer, regarding the printing sequence and the way the printer uses its sensors, we had to record its actions while printing normal ink on normal paper. We found easy to trick the lid sensor, but without removing any of the functional components the paper feed sensor is quite tricky to overpass. The next milestone for this part of the project is to determine:
The modification on the printer body. Whether its to be kept or discarded for a custom tray feed mechanism
The means of tricking the paper feed sensor, either by using mechanical methods or electronic methods
What is decided:
The printer body is to be kept. We are going to use the main structure of the printer but remove excess parts as the head cleaning tray, the paper transport tray and some axis. Although we don't directly need the main paper feed motor, we are going to keep it because it helps tricking the feed sensor. Concerning that, we are going to use an Arduino micro-controller. The controller will get signal from the main motor. We found that when the paper sensor is enabled, the motor change rotation for an instant. In correlation to the fact that the motor is DC powered, we are using that change in the voltage (shown in the right picture) to signal the controller. In its turn, the controller turns on an external infrared led emitter (the appropriate voltage required was measured, shown in the left picture) which signals the sensor instead of his own (blocked) emitter. In that way, we use the original circuit of the printer without any internal changes, which guarantees its function.
Motor voltage (oscillograph monitor)
Sensor emitter voltage (Oscillograph monitor)
Milestone 3: Prototype fabrication - Experiments
Biological Experiments :
The following experiments are considered important to determine the survivability of cells through the whole procedure (bio-ink fabrication, storage in ink cartridge, printing):
Test type
Cell Type
Results
1 ) Test viability of cell sample
Yeast
Success
2 ) Test viability after printing
Yeast
Success
3 ) Test viability after adding printing enhancer (glycerol, ...)
Yeast
Not needed**
4 ) Test viability of cell sample
Other*
Success
5 ) Test viability after remaining for 20 minutes in ink cartridge
Other
Success
6 ) Test viability after printing
Other
Success
7 ) Find bio-compatible ways to regulate bio-ink's viscosity
All
Not needed
Other cell types must be declared (fiborblasts, chondrocytes, cartilage tissue)
The "Not needed" indicates that the printing is successful without the need to regulate the bio-ink's viscosity. The viscosity regulation presupposes a reliable way to measure it as well as a reliable method to calculate the cell's density in the ink. Both measurements could be done with great difficultly or not at all in a cell solution.
Results
Cell type
=====
Yeast
==========
Fibroblast
==================
Live cells before printing
119
25
Dead cells before printing
28
13
Viability before printing
============
80%
65%
Live cells after printing
106
17
Dead cells after printing
77
42
Viability after printing
============
60%
30%
Total Survivability
(through printing process)
75%
50%
In the beginning we experimented with the yeast samples, because yeast is smaller dimension cell, so the printing would be easier to happen. The process was successful and here lie the printed sample:
The following images are displaying the results of the experiments 5,6. We tried two tests with the same cells (fibroblasts). On the left there are the control samples while on the right you can observe the printed cells. Both of the samples are colored with trypan blue in order for the mortality of the printing to be measured.
Fibroblast cells before printing 1 (Control sample 1)
Fibroblast cells after printing 1 (Test sample 1)
Fibroblast cells before printing 2 (Control sample 2)
Fibroblast cells after printing 2 (Test sample 2)
Yeast before printing
Yeast after printing
Future prospects
This project surely has the potential to constitute the base for future works concerning bio-printing with cheap and easy to find equipment. There are still a number of issues that has not been investigated in the current project. Such issues that can be solved in the near future are:
• Construction of the appropriate cover in order to avoid direct contact with specific parts of the printer (mostly for safety reasons)
• Manually adjustable printing-stage (so as to print on different surfaces)
Some more sophisticated future directions concerning this project are:
• Construction of an automatically adjustable moving stage in y-axis (paper-feed axis)
• Construction of an automatically adjustable moving stage in z-axis. Ability to print 3D bio-components
• Automation of the pre-printing procedure (e.g. clean cartridge, add cell-sample to cartridge, adjust petri-dish to stage)
• Automation of the post-printing procedure (e.g. transfer petri-dish to microscope, examine cell-sample)
(c) 2015 Department of Mechanical Engineering, National Technical University of Athens. 1. Inkjet printing of viable mammalian cells: http:www.sciencedirect.com/science/article/pii/S01429612040033572. Printing technologies for biomolecule and cell-based applications: http:www.sciencedirect.com/science/article/pii/S0378517315001337?np=y3. Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology http:www.jove.com/video/51294/human-cartilage-tissue-fabrication-using-three-dimensional-inkjet4. Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer http:www.jove.com/video/3681/creating-transient-cell-membrane-pores-using-a-standard-inkjet-printer5.Inkjet printing for high-throughput cell patterning:http:www.sciencedirect.com/science/article/pii/S01429612030100936.Drop-on demand inkjet bioprinting: a primer:http:
www.worldscientific.com/doi/abs/10.1142/s1568558611000258 Web sites2.1http://store.hp.com/UKStore/Merch/Product.aspx?id=51626AE&opt=&sel=SUP2.2http://nerdcreationlab.com/Store/BIOPRINTNG
Project Info
Objective:Modify a standard commercially available inkjet printer in order to print cells or biopolymer solutions.
Team Members:
1. Αναγνώστου Γεώργιος
2. Διανέλλος Γεώργιος
3. Ρόβας Γιώργος
4. Ρούβαλης Νικόλαος
Team Contact:
Rouvalis Nikolaos (mc11016@mail.ntua.gr)
Mentor:
Polesiouk Alexander (alexanderpol3@gmail.com)
Consultants:
Tzeranis Dimitrios (tzeranis@gmail.com)
Sarkiri Maro (marw.sarkiri@hotmail.com)
Project-report:
Arduino-code:
Milestone 1: Preliminary design / State of the art
State of the art
Along with the development of 2D/3D printed polymers, there have been efforts for printing living cells or other organic molecules. There are various methods used, but the one of interest for the current project is modifying an inkjet printer into a bio/cell printer. Multiple scientists around the world have successfully modified commercial thermal and piezoelectric inkjet printers to accomplish this task [3]. The type of printed molecules vary from proteins or other organic molecules to mammalian living cells.
Drop-on-demand bioprinting is commonly used with modified desktop inkjet printers. Modification of desktop printers is both an advantage and disadvantage of this technique. The hardware interfacing with the printhead already exists and has been quality tested by the manufacturer. However, this hardware is designed to print on paper and contains numerous checks to prevent the printer from performing outside its normal specifications. Depending on the bioprinting application, the printer hardware must either be reverse-engineered or modified to bioprint. A printer can be modified to bioprint by using a bioprinting stage on one side of the printer and feeding paper through the other side to bypass the paper checks. Reverse engineering provides control of individual inkjet channels which is extremely beneficial for studying the fundamentals of inkjet bioprinting, especially with regard to channel throughput and clogging. However, reverse engineering a desktop printer may be beyond the capabilities of research groups that do not focus on engineering. Therefore, several groups have used the existing printer hardware with modifications to facilitate bioprinting.
One of the major modifications made to desktop inkjet printers is the addition of a Z axis in the paper tray. Since a desktop printer only prints two-dimensional sheets of paper, stacking multiple layers at once requires an additional mechanism to lower the printing area with each successive layer. This is typically performed by adding an electronic elevator to the paper tray that lowers upon receiving a signal from the operator to print another layer. In this way, additional layers can be stacked upon each other to create a three-dimensional construct from two-dimensional layers.
Despite the numerous applications of inkjet bioprinting, there are two major issues that prevent single-cell inkjet bioprinting from reaching its full potential. The first issue is the limited mobility of the printhead if no reverse engineering is performed. In the normal configuration the printhead is limited to less than 250mm on the X axis and the length of the printhead on the Y axis. Thus, only biological structures of limited size can be created with this type of bioprinting system. Second, inkjet cartridges suffer from low throughput. This is mainly due to deposition of salts in the microfluidic channels during the printing process. This often occurs when evaporation of water from the bio-ink drop leaves behind solid salts that block the channel orifice. Once the channel is clogged, it is virtually impossible to restore full functionality to that channel. Furthermore, cellular debris and other contaminants can clog the microfluidic chambers as well. As a result, inkjet cartridges typically can only print 400,000 cells per cartridge before failure. This throughput is too low to produce large tissue constructs.
It should be noted that older printers tend to produce better results after the modification, as they use ink cartridges with larger diameter nozzles, which do not clog easily and allow to print larger cells. In addition, older printers use mechanical paper feed sensors that are easier to bypass. (Printers with optical sensors can also be “tricked”). Similar attempts have been made using HP DeskJet 500 [4]. Each application of bioprinting requires a separate set of cell types and matrices. Studies of cell viability in thermal inkjet printheads have shown cell survival to be 70–90%, although some groups have determined that cells require a recovery period after bioprinting to restore membrane integrity.
Design Specifications
Ink Cartridge specifications:
Black: No. 339 (21 ml)
Drop: 15 picoliters
Nozzles: 672
Nozzle diameter:
Head Length: 0.5625 in
DPI : max 1200 , fast 300, normal 600
Color: No. 343 (7 ml per color)
Drop: 5 picoleters
Nozzles: 600
Nozzle diameter ~= 10μm approx
Head Length: 0.5 in
DPI: max 1200, fast 300, normal 600
=====================================
Ink Cartridge specifications:
Black: No. 15 (21 ml)
Drop: 33 picoliters
Nozzles: N/A
Nozzle diameter: ~35 μm (measured, not provided)
Head Length: ~0.5 in
DPI : max 600 , normal 300
Color: No. 17 (7 ml per color)
Drop: 9 picoliters
Nozzles: N/A
Nozzle diameter N/A (not measured, not provided)
Head Length: N/A
DPI: max 1200, normal 600
================================
SELECTED
Key Functions
Protocol for modifying the ink cartridge:
Use any long tool with flat edge, for example a straight screwdriver (img 3 for explanations)
Now your ink cartridge is ready to be cleaned and sterilized.
Protocol for cleaning ink cartridge:
Protocol for cleaning the printer:
Protocol for printing samples:
Milestone 2: Detailed design
Reverse engineering of a similar printer
We have deconstructed an Officejet J6400 All-in-One printer in order to discover the key mechanisms of the device, and which of those will or could be used for the task we want to accomplish. Both the J6400 and the D4260 use the same ink cartridges and it is quite possible to have the same mechanisms and control units. What we found is that the different mechanisms are:
1) Ink cartridges/print head holder
2) Paper feeding mechanism
3) Print head dust/air contact protection mechanism
4) Print head cleaning/unclogging mechanism
5) Paper feed sensor (mechanical lever & opto switch) (easy to bypass)
6) Lid status (open/close) sensor (mechanical lever & opto switch) (easy to bypass)
7) Paper and Print head carriage motors
8) Paper motor and print head carriage position feedback and sensors
Print head size & structure using microscope for D4260
In order to determine the size of cells that can be "printed" using the Deskjet D4260 printer, it is essential to know the diameter of the nozzles in each of the ink cartridges (blank/color). To do so, we disassembled the print head of an ink cartridge (typically a thin, silver foil with the nozzle holes and ink channels drilled in it) and examined it using two different microscopes.
According to the 3rd picture, the nozzle diameter is about 10μm with a 80μm spacing between nozzles. Only the tri-color ink was disassembled and inspected in detail. The nozzles of the black ink are expected to be slightly bigger because its drop volume is three times larger than the one of the tri-color ink (see ink specs.), but still it should not exceed 15μm. This allows only the printing of bio-molecules, prokaryotic cells and only small eukaryotic cells with average cell diameter roughly equal to the one of the nozzle.
Ink cartridges contain a sponge which absorbs the main volume of the ink. Between this sponge and the channels that lead the ink to the printing nozzles are placed various filters to keep solid particles from entering and blocking the nozzles. The pore diameter of the main sponge is smaller than 50μm, while that of the filters is way smaller than 10μm. These pores do not allow the ink cartridge to be filled with a cell solution, because most cells can not pass through and the filters will be quickly blocked. Furthermore, cleaning and sanitizing the cartridge is more difficult if it contains filters. As a result, both the sponge and the filters must be removed, before any other process is initiated.
Another important parameter is the minimum volume of bio-ink required. If the volume of ink is smaller that this quantity, the pressure generated from the inks own weight is not enough to overcame the hydraulic resistance of the channels. As a result, the ink never reaches the nozzles and the printing has very low quality or is not done at all. This parameter can only be measured by experimentation. For this printer its value is 0.5 ml.
Print head size & structure using microscope for 825c
The measurement was repeated for the black ink of the DeskJet 825c printer, which was also available for experimentation. As we can see on the left image, the nozzle diameter is 35μm, three times larger than that of the D4260, thus allowing the printing of much larger cells. The distance between successive nozzles is about 50μm with an 10μm offset from the straight, central line.
As for the minimum volume of ink of this printer, it was measured with experiments and it is 0.25 ml.
Other possible printers - Final Decision
The nozzle diameter of the printer's cartridges disallows most eukaryotic cells to be printed without serious stress. So we had to find printers that use older cartridges with larger nozzles. One of the most suitable options is the HP 26 Black Ink Cartridge [2.1] with a drop volume of 140-150 picoliters (>10 times larger than the HP 339 Ink) and a nozzle diameter of about 50μm. Unfortunately, most printers that use this cartridge are not sold today. Although, there are some used ones available but their price ( ~90$) exceeds the budget of this project. Additionally, it is unclear if these printers will still work during the lifetime of the project and whether spare parts and cartridges will be marketable the following years.
There are also some custom made print heads, usually controlled by an arduino device. They offer very low DPI values and accordingly large nozzle diameters. One of these is the InkShield with 96x96 DPI and nozzle diameter equal to 85μm, features that make it ideal for the purpose. The price of an InkShield DIY Kit is about 60$ [2.2], which is too beyond the project's budget.
To summarize, there are some good but limited options available in the market, but their price is the main reason we will choose to continue working with an already owned printer and setting an upper limit on the diameter of cells that can be printed. Both printers, D4260 and 825c, were reversed engineered and reached a level, where they could be manually operated to print cells or biomolecules. It was important, though, to choose only one of them for further experiments, automatize its printing process and modify it, until it reaches the desired design level. The main factor that affected this choice was the nozzle diameter and the range of cells that can be printed. Thus, the DeskJet 825c is considered, as previously mentioned, a superior choice, with the following advantages:
Sensors and systems to be tricked / used
Determining the sequence of functions during the printing
In order to modify the printer, regarding the printing sequence and the way the printer uses its sensors, we had to record its actions while printing normal ink on normal paper. We found easy to trick the lid sensor, but without removing any of the functional components the paper feed sensor is quite tricky to overpass. The next milestone for this part of the project is to determine:
What is decided:
The printer body is to be kept. We are going to use the main structure of the printer but remove excess parts as the head cleaning tray, the paper transport tray and some axis. Although we don't directly need the main paper feed motor, we are going to keep it because it helps tricking the feed sensor. Concerning that, we are going to use an Arduino micro-controller. The controller will get signal from the main motor. We found that when the paper sensor is enabled, the motor change rotation for an instant. In correlation to the fact that the motor is DC powered, we are using that change in the voltage (shown in the right picture) to signal the controller. In its turn, the controller turns on an external infrared led emitter (the appropriate voltage required was measured, shown in the left picture) which signals the sensor instead of his own (blocked) emitter. In that way, we use the original circuit of the printer without any internal changes, which guarantees its function.
Milestone 3: Prototype fabrication - Experiments
Biological Experiments :
The following experiments are considered important to determine the survivability of cells through the whole procedure (bio-ink fabrication, storage in ink cartridge, printing):
=====
==========
==================
============
============
(through printing process)
In the beginning we experimented with the yeast samples, because yeast is smaller dimension cell, so the printing would be easier to happen. The process was successful and here lie the printed sample:
The following images are displaying the results of the experiments 5,6. We tried two tests with the same cells (fibroblasts). On the left there are the control samples while on the right you can observe the printed cells. Both of the samples are colored with trypan blue in order for the mortality of the printing to be measured.
Future prospects
This project surely has the potential to constitute the base for future works concerning bio-printing with cheap and easy to find equipment. There are still a number of issues that has not been investigated in the current project. Such issues that can be solved in the near future are:
• Construction of the appropriate cover in order to avoid direct contact with specific parts of the printer (mostly for safety reasons)
• Manually adjustable printing-stage (so as to print on different surfaces)
Some more sophisticated future directions concerning this project are:
• Construction of an automatically adjustable moving stage in y-axis (paper-feed axis)
• Construction of an automatically adjustable moving stage in z-axis. Ability to print 3D bio-components
• Automation of the pre-printing procedure (e.g. clean cartridge, add cell-sample to cartridge, adjust petri-dish to stage)
• Automation of the post-printing procedure (e.g. transfer petri-dish to microscope, examine cell-sample)
=
References
Research Papers
1.Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology
http://www.jove.com/video/51294/human-cartilage-tissue-fabrication-using-three-dimensional-inkjet
2. Inkjet printing of viable mammalian cells:
http://www.sciencedirect.com/science/article/pii/S01429612040033573. Printing technologies for biomolecule and cell-based applications:
http://www.sciencedirect.com/science/article/pii/S0378517315001337?np=y4.Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer
http://www.jove.com/video/3681/creating-transient-cell-membrane-pores-using-a-standard-inkjet-printer
5. Inkjet printing for high-throughput cell patterning:
http://www.sciencedirect.com/science/article/pii/S0142961203010093
6.Drop-on demand inkjet bioprinting: a primer
http://www.worldscientific.com/doi/abs/10.1142/s1568558611000258
Web sites
2.1
http://store.hp.com/UKStore/Merch/Product.aspx?id=51626AE&opt=&sel=SUP
2.2
http://nerdcreationlab.com/Store/
(c) 2015 Department of Mechanical Engineering, National Technical University of Athens.
1. Inkjet printing of viable mammalian cells: http:www.sciencedirect.com/science/article/pii/S01429612040033572. Printing technologies for biomolecule and cell-based applications: http:www.sciencedirect.com/science/article/pii/S0378517315001337?np=y3. Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology http:www.jove.com/video/51294/human-cartilage-tissue-fabrication-using-three-dimensional-inkjet4. Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer http:www.jove.com/video/3681/creating-transient-cell-membrane-pores-using-a-standard-inkjet-printer5.Inkjet printing for high-throughput cell patterning:http:www.sciencedirect.com/science/article/pii/S01429612030100936.Drop-on demand inkjet bioprinting: a primer:http:
www.worldscientific.com/doi/abs/10.1142/s1568558611000258
Web sites2.1http://store.hp.com/UKStore/Merch/Product.aspx?id=51626AE&opt=&sel=SUP2.2http://nerdcreationlab.com/Store/BIOPRINTNG